The present invention relates to phosphonic acid derivatives useful in the treatment of cardiovascular diseases and, more particularly, it relates to phosphonic acid derivatives useful in the treatment of cardiovascular diseases as metallopeptidase inhibitors. The pharmacologic interest towards the study of metallopeptidase inhibitory molecules derives from the role that said enzymes exert on the level of the cardiocirculatory system.
It is well-known in fact that compounds with angiotensin converting enzyme (ACE) inhibitory activity are mainly useful in the treatment of hypertension, of heart failure and of post-infarct in that they inhibit the formation of angiotensin II, a substance which increases the blood pressure.
Compounds with endothelin converting enzyme (ECE) inhibitory activity are useful as anti-vasoconstrictors in that they inhibit the formation of endothelin, a 21 amino acid peptide with vasoconstrictor activity.
Instead, compounds with inhibitory activity of the neutral endopeptidase (NEP) enzyme, also called enkephalinase, are useful as vasodilators and diuretics in that the NEP enzyme is responsible for the inactivation, not only of endogenous enkephahne, but also of some natriuretic factors among which, for instance, the atrial natriuretic factor (ANF), a vasodilating hormone secreted by heart.
Therefore, even exerting their action on the cardiovascular system with different mechanisms of action, the compounds with metallopeptidase inhibitory activity are generally used, alone or in combination, in the treatment of hypertension, renal failure, congestive heart failure and post-infarct.
In the U.S. Pat. No. 4,432,972 (E.R. Squibb and Sons, Inc.) phosphorylated derivatives of amino acids such as, in particular, phosphonamidates endowed with ACE-inhibitory and enkephahnase-inhibitory activity were described.
Said compounds were described as useful hypotensive and analgesic agents. In the European patent application No. 0518299 (Takeda Chemical Industries, Ltd) some phosphonic acid derivatives, endowed with ECE-inhibitory activity, usefull in the treatment of hypertension, of cardiac or cerebrovascular diseases and of renal diseases were described.
Now we have found phosphonic acid derivatives which are endowed with inhibitory activity on the angiotensin converting enzyme as well as on the neutral endopeptidase enzyme (dual ACE/NEP-inhibitory activity) which renders them particularly useful in the cardiovascular therapy.
Therefore object of the present invention are the compounds of formula 
wherein
R is a straight or branched C1-C6 alkyl group optionally substituted with one or more fluorine atoms, an aryl or arylalkyl group with from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl 1-naphthyl, 2-naphthyl group or a 5 or 6 membered aromatic heterocycle with 1 or 2 heteroatoms selected among nitrogen, oxygen and sulphur, optionally substituted with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkyl, alkoxy, alkythio, alkylsulphonyl or alkoxycarbonyl groups with from 1 to 3 carbon atoms in the alkyl moiety, carboxy groups, aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylaminocarbonyl groups with from 1 to 3 carbon atoms in the alkyl moiety;
R1 and R2, the same or different, represent a hydrogen atom or a straight or branched C1-C4 alkyl group;
R3 is a straight or branched C1-C6 alkyl group or an arylalkyl group with from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, 1-naphthyl 2-naphthyl group or a 5 or 6 membered aromatic heterocycle with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substituted as indicated for R;
R4 is a 5 or 6 membered aromatic heterocyclic group with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substituted with a 5 or 6 membered aromatic heterocyclic group with one or two heteroatoms selected among nitrogen, oxygen and sulphur or with a phenyl group, or it is a phenyl group substituted with a 5 or 6 membered aromatic heterocyclic group with one or two heteroatoms selected among nitrogen, oxygen and sulphur, being the phenyl and the heterocyclic groups optionally substituted with one or more substituents, the same or different, selected among halogen atoms, alkyl alkoxy, alkylthio or alkoxycarbonyl groups with from 1 to 3 carbon atoms in the alkyl moiety;
X is a single bond or an xe2x80x94Oxe2x80x94CONHxe2x80x94 or xe2x80x94CONHxe2x80x94 group;
the carbon atoms marked with an asterisk are asymmetric carbon atoms;
and pharmaceutically acceptable salts thereof;
provided that R4 is not an imidazolyl or indolyl group.
The compounds of formula I contain at least two asymmetric carbon atoms and can thus exist in the form of stereoisomers.
Therefore, object of the present invention are the compounds of formula I in the form of stereoisomeric mixture as well as in the form of single stereoisomers.
The compounds of formula I object of the present invention are endowed with a dual ACE/NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases.
In the present description, unless otherwise specified, with the term alkyl group we intend a straight or branched alkyl such as methyl, ethyl, n.propyl, isopropyl, n.butyl, sec-butyl tert-butyl isobutyl n.pentyl 2-pentyl, 3-pentyl, isopentyl, tert-pentyl n.hexyl and isohexyl; with the term alkoxy group we intend a straight or branched alkoxy such as methoxy, ethoxy, n.propoxy and isopropoxy; with the term halogen atom we intend a fluorine, chlorine, bromine or iodine atom; with the term acyl we intend an acyl group deriving from an aliphatic or aromatic carboxylic acid such as acetic, propionic, butyric and benzoic acid; with the term aryl we intend an aromatic group such as phenyl, 1-naphthyl, 2-naphthyl or a 5 or 6 membered heterocyclic group containing 1 or 2 heteroatoms selected among nitrogen, oxygen and sulphur such as thiazole, isoxazole, oxazole, isothiazole, pyrazole, imidazole, thiophene, pyrrole, pyridine, pyrimidine, pyrazine and furan, optionally benzocondensed.
Examples of pharmaceutically acceptable salts of the compounds of formula I are the salts with alkali or alkali-earth metals and the salts with pharmaceutically acceptable organic bases.
Preferred compounds of formula I are the compounds wherein R4 represents a phenyl group substituted in position 4 with a heterocyclic group.
Particularly preferred, in this class, are the compounds of formula I wherein R1 and R2 represent a hydrogen atom and R3 represents a straight or branched C1-C4 alkyl group.
Preferred examples of pharmaceutically acceptable salts of the compounds of formula I are the salts with alkali metals such as sodium, lithium and potassium.
Specific examples of preferred compounds of formula I, object of the present invention, are:
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(2-furyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(3-furyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(2-thienyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(3-thienyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanne;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(2-thiazolyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(2-pyridyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(3-pyridyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-(4-pyrazinyl)-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-leucyl)-[4-(5-pyrmidinyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(2-furyl)]-phenylalaine;
N-(Nxe2x80x2-propyhphosphonyl-valyl)-[4-(3-furyl)]-phenylalanine;
N-(Nxe2x80x2-propyhphosphonyl-valyl)-[4-(2-thienyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(3-thienyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(2-thiazolyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(2-pyridyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(3-pyridyl)]-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)(4-pyrazinyl)-phenylalanine;
N-(Nxe2x80x2-propylphosphonyl-valyl)-[4-(5-pyrimidinyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-furyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(3-fiuyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(3-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-thiazolyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(2-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(3-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-(4-pyrazinyl)-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-leucyl]-[4-(5-pyrimidinyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(2-furyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(3-furyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienyhnethyl)phosphonyl-valyl]-[4-(2-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(3-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(2-thiazolyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(2-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(3-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(2-thienyhmethyl)phosphonyl-valyl]-(4-pyrazinyl)-phenylalanine;
N-[Nxe2x80x2-(2-thienylmethyl)phosphonyl-valyl]-[4-(5-pyrmidinyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-furyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(3-furyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(3-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl)-[4-(Nxe2x80x3methyl-2-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-thiazolyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(2-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(3-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-(4-pyrazinyl)-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-leucyl]-[4-(5-pyrimidinyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-furyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(3-furyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(3-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-thiazolyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(2-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(3-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-(4-pyrazinyl)-phenylalanine;
N-[Nxe2x80x2-(benzyloxycarbonylaminomethyl)phosphonyl-valyl]-[4-(5-pyrimidinyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-furyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(3-furyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(3-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-thiazolyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(2-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(3-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-(4-pyrazinyl)-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-leucyl]-[4-(5-pyrimidinyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-furyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(3-furyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(3-thienyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(Nxe2x80x3-methyl-2-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(Nxe2x80x3-methyl-3-pyrrolyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-thiazolyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(2-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(3-pyridyl)]-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-(4-pyrazinyl)-phenylalanine;
N-[Nxe2x80x2-(acetylaminomethyl)phosphonyl-valyl]-[4-(5-pyrimidinyl)]-phenylalanine.
The preparation of the compounds of formula I, object of the present invention, comprises the reaction between a phosphorylated derivative of formula 
wherein
R, R1 and X have the above reported meanings, W represents a halogen atom, preferably chlorine, and Y represents a protective group, preferably a C1-C4 alkyl, a phenyl or a phenylalkyl with from 1 to 4 carbon atoms in the alkyl moiety;
and a dipeptide derivative of formula 
xe2x80x83wherein
R2, R3 and R4 have the above reported meanings.
The phosphorylated derivative of formula II can be prepared from the corresponding compound of formula 
wherein
R, R1 and X have the above reported meanings and the Z groups both represent a W or OY group wherein W and Y have the above reported meanings.
The preparation of the compounds of formula II from the corresponding compounds of formula IV is carried out according to conventional techniques, by reaction with a halogenating agent or with a compound of formula YOH, wherein Y has the above reported meanings.
For a reference to the preparation of the compounds of formula II wherein X is a single bond or an xe2x80x94Oxe2x80x94CONHxe2x80x94 group see, for instance, D. S. Karanewsky et at, J. Med. Chem. 1988, 31, 204-212 and B. P. Morgan et al, J. Am Chem. Soc. 1991, 113, 297-307.
The compounds of formula II wherein X is a xe2x80x94CONHxe2x80x94 group can be prepared, as example, from the corresponding compounds of formula II wherein X is an xe2x80x94Oxe2x80x94CONHxe2x80x94 group and R=benzyl through hydrogenolysis of the carbamic group (Rxe2x80x94Oxe2x80x94CONHxe2x80x94) and subsequent reaction with a compound of formula
Rxe2x80x94COW1xe2x80x83xe2x80x83(V)
wherein
R has the above reported meanings and W1 represents a chlorine or bromine atom.
The dipeptide derivatives of formula III, in their turn, can be prepared through the condensation between an amino acid of formula 
wherein
R2 and R3 have the above reported meanings;
and an alanine derivative of formula 
xe2x80x83wherein R4 has the above reported meanings.
The condensation reaction is carried out according to conventional techniques of the chemistry of peptides.
Before carrying out the reaction it can be useful to properly protect the optional functional groups which could interfere in the reaction.
The optional protection is carried out according to conventional techniques.
For instance, in the reaction between the phosphorylated derivative of formula II and the dipeptide derivative of formula III it can be useful to protect the free carboxy function of the compound of formula III as well as the hydroxy function of the phosphonic group.
Likewise, in the reaction between the amino acid of formula VI and the alanine derivative of formula VII, it can be useful to protect the amino function of the derivative of formula VI and the carboxy function of the derivative of formula VII.
The evaluation of the usefulness of the optional protection as well as the selection of the kind of adopted protection according to the reaction to be carried out and to the functional groups to be protected are within the normal knowledge of the man skilled in the art.
The removal of the optional protective groups is carried out according to conventional techniques.
For a general reference to the use of protective groups in organic chemistry see Theodora W. Greene and Peter G. M. Wuts xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, John Wiley and Sons, Inc., II Ed., 1991.
The compounds of formula VI and VII are known or easily prepared according to known methods.
For a reference to the preparation of the compounds of formula VII see, for instance, the synthetic methods described by W. C. Shieh and T. R. Bailey in J. Org. Chem. 1992, 57, 379-381 and Tetrahedron Letters, 27, 4407-4410, 1986, respectively.
The compounds of formula I, object of the present invention, can be further prepared according to an alternative synthetic scheme comprising the reaction between a phosphorylated derivative of formula 
wherein
R, R1, R2, R3, X and Y have the above reported meanings;
and an alanine derivative of formula VII.
Analogously to what previously reported, also in the above reaction it can be useful to protect, according to conventional techniques, eventual functional groups which could interfere in the reaction.
The compounds of formula VIII are known or easily prepared according to known methods.
For instance, the compounds of formula VIII can be prepared through the reaction between the phosphorylated derivative of formula II and the amino acid of formula VI according to conventional methods of the chemistry of peptides.
In view of what above indicated, it is clear to the man skilled in the art that the preparation of the compounds of formula I wherein X is a xe2x80x94CONHxe2x80x94 group can be optionally carried out starting from the corresponding compounds of formula I wherein X is an xe2x80x94Oxe2x80x94CONHxe2x80x94 group and R=benzyl, prepared according to one of the aforementioned synthetic methods.
The compounds of formula I in the form of single stereoisomers are prepared by stereo selective synthesis or by separation of the stereoisomeric mixture according to conventional techniques.
Also the preparation of the salts of the compounds of formula I, object of the invention, is carried out according to conventional techniques.
The compounds of formula I object of the present invention are endowed with a dual ACE/NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases.
The inhibitory activity of the compounds of formula I, in particular, was evaluated by means of in vitro and ex vivo tests.
The in vitro inhibitory activity of the compounds of formula I was evaluated in comparison to known molecules endowed with ACE-inhibitory or NEP-inhibitory activity (example 3).
Captopril, a drug known as the first orally active ACE-inhibitor (The Merck Index, XI ed.xe2x80x94No. 1773, pages 267-268), was used as a comparison compound for the ACE-inhibitory activity.
Thiorphan [DL-(3-mercapto-2-benzylpropionyl)glycine] instead, known molecule considered the parent compound for NEP-inhibitors and described for the first time by Roques et al. in Nature, vol. 288, pages 286-288, (1980), was used as a comparison compound for the NEP-inhibitory activity.
N-[Nxe2x80x2-(4-phenyl)butylphosphonyl-L-phenylalanyl]-L-phenylalanine dilithium salt (hereinafter referred to as compound R-1), exemplified in the aforementioned U.S. Pat. No. 4,432,972, was considered as a further comparison compound for evaluating the in vitro inhibitory activity of the compounds of formula I.
The in vitro inhibitory activity of the compounds of formula I, expressed as IC50 value, is pharmacologically significant in that it results at nM concentrations.
Said activity resulted to be at least comparable to that of Captopril, to what it concerns the ACE-inhibitory activity, and to that of Thiorphan, to what it concerns the NEP-inhibitory activity.
With respect to the compound R-1, moreover, the dual ACE/NEP-inhibitory activity of the compounds of formula I resulted to be significantly higher.
As above indicated, the inhibitory activity of the compounds of formula I was evaluated also by means of ex vivo experiments, using the aforementioned compound R-1 as a comparison compound (example 4).
The dual ACE/NEP-inhibitory activity ex vivo of the compounds of formula I resulted to be significantly higher than that of the comparison compound.
For a practical use in therapy, the compounds of formula I can be formulated in solid or liquid pharmaceutical compositions, suitable to oral or parenteral administration.
Therefore, the pharmaceutical compositions containing a therapeutically effective amount of a compound of formula I in admixture with a carrier for pharmaceutical use are a further object of the present invention.
Specific examples of pharmaceutical compositions according to the present invention are tablets, coated tablets, capsules, granulates, solutions and suspensions suitable to oral administration, solutions and suspensions suitable to parenteral administration.
The pharmaceutical compositions object of the present invention are prepared according to conventional techniques.
Although the compounds of formula I are active as such, with the aim to satisfy particular therapeutic or pharmaceutical properties, it can be useful to transform them into the corresponding biologic precursors (pro-drugs).
Therefore, according to the conventional techniques for the preparation of pro-drugs of phosphorylated and amido derivatives, suitable pro-drugs can be obtained for instance through the esterification of the carboxy function or of the phosphonic function.
Also the compounds of formula I in the form of pro-drugs and, in particular, the compounds obtained through the esterification of the carboxy or phosphonic function, as well as the pharmaceutical compositions which contain the compounds of formula I in the form of pro-drugs and, in particular, which contain the compounds of formula I wherein the carboxy or phosphonic group results to be esterified, are within the scope of the present invention.
The daily dose of the compound of formula I or of the corresponding pro-drug will depend on several factors such as the seriousness of the disease, the individual response of the patient or the kind of formulation but it is usually comprised between 0.01 mg and 20 mg per kg of body weight divided into a single dose or into more daily doses.